PROJECT SUMMARY Millions of patients worldwide undergo general anesthesia each year. However, despite ubiquitous use in medical practice, the molecular mechanisms by which general anesthetics cause immobilization, unconsciousness, amnesia, and analgesia remain elusive. The objective of this proposal is to provide mechanistic insight into the molecular basis of general anesthesia for the design of safer anesthetic agents. It is widely believed that general anesthetics interact directly with proteins of the central nervous system. Because voltage-gated Na+ channels (Navs) play a pivotal role in the electrical excitability of the nervous system, they represent a class of highly relevant targets. All commonly used general anesthetics inhibit Navs, but the biophysical and structural bases of this inhibition remain poorly understood. We hypothesize that general anesthetics inhibit Navs by interacting with binding sites allosterically coupled to regions that control slow inactivation gating. This proposal studies NaChBac and NavMs, prokaryotic homologs and surrogates of eukaryotic Navs, to gain fundamental insight into the mechanism of Nav inhibition by propofol, the most commonly used intravenous general anesthetic. The aims of this project are: (1) to investigate the molecular determinants of Nav inhibition by propofol and (2) to investigate propofol binding in Navs with photoaffinity labeling. To accomplish these aims, we will use a combination of patch-clamp electrophysiology, molecular dynamics simulations, mutational analysis, protein biochemistry, and photoaffinity labeling. This diverse array of complementary techniques will allow us to identify sites of propofol action in Navs and elucidate a mechanism of action. Completion of this work will lay a foundation for future structural investigations and the rational design of safer general anesthetic agents.